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DMD #58107
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TITLE PAGE
In Vitro Oxidative Metabolism of 6-Mercaptopurine in Human Liver: Insights into the
Role of the Molybdoflavoenzymes, Aldehyde Oxidase, Xanthine Oxidase and Xanthine
Dehydrogenase.
Kanika V. Choughule, Carlo Barnaba, Carolyn A. Joswig-Jones, Jeffrey P. Jones
Department of Chemistry, Washington State University, Pullman, Washington
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Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title – Oxidative metabolism of 6-Mercaptopurine
Address correspondence to: Jeffrey P. Jones, Department of Chemistry, Washington State
University, and P.O. Box 644630, Pullman, Washington 99164-4630. Email: [email protected]
Document Summary
Text Pages 16
Tables 3
Figures 5
References 29
Word Count:
Abstract: 241
Introduction: 764
Results and Discussion: 1,409
Abbreviations – 6MP, 6-mercaptopurine; 6TX, 6-thioxanthine; 6TUA, 6-thiouric acid; AO,
Aldehyde oxidase; XO, Xanthine oxidase; HLC, human liver cytosol; XOR, Xanthine
oxidoreductase; XDH, Xanthine dehydrogenase ; ALL, Acute lymphoblastic leukemia;
TIMP, thioinosinic acid; HGPRtase, hypoxanthine-guanine phosphoribosyltransferase; 6-
TGN, 6-thioguanine; 8-oxo-6MP, 8-oxo-6-mercaptopurine; 6-Me-8OH-MP, 6-
methylmercapto-8-hydroxypurine; MeMP, Methylmercaptopurine; IPTG, Isopropyl β-D-
thiogalactoside; AOX1, gene encoding human aldehyde oxidase; NAD, Nicotinamide
adenine dinucleotide
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ABSTRACT
Anti cancer agent, 6-mercaptopurine (6MP) has been in use since 1953 for the treatment of
childhood acute lymphoblastic leukemia (ALL) and inflammatory bowel disease. Despite
being around for 60 years, several aspects of its drug metabolism and pharmacokinetics in
human are unknown. Molybdoflavoenzymes such as aldehyde oxidase (AO) and xanthine
oxidase (XO) have previously been implicated in the metabolism of this drug. In this study,
we investigated the in vitro metabolism of 6MP to 6-thiouric acid (6TUA) in pooled human
liver cytosol. We discovered that 6MP is metabolized to 6TUA through sequential
metabolism via the 6-thioxanthine (6TX) intermediate. The role of human AO and XO in the
metabolism of 6MP was established using specific inhibitors raloxifene and febuxostat. Both
AO and XO were involved in the metabolism of the 6TX intermediate whereas only XO was
responsible for the conversion of 6TX to 6TUA. These findings were further confirmed using
purified human AO and E. coli lysate containing expressed recombinant human XO.
Xanthine dehydrogenase (XDH) which belongs to the family of xanthine oxidoreductases and
preferentially reduces NAD+ was shown to contribute to the overall production of the 6TX
intermediate as well as the final product 6TUA in the presence of NAD+ in human liver
cytosol. In conclusion, we present evidence that three enzymes, AO, XO and XDH,
contribute to the production of 6TX intermediate whereas only XO and XDH are involved in
the conversion of 6TX to 6TUA in pooled HLC.
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INTRODUCTION
6-mercaptopurine (6MP) is a thiopurine drug with anti-tumor activity that has been in use as
a remission inducing agent for the treatment of childhood acute lymphoblastic leukemia
(Burchenal et al., 1953). It has also been used as an immunosuppressive agent in combination
with its prodrug, azathioprine for the treatment of inflammatory bowel disease such as
ulcerative colitis and Crohn’s disease (Nielsen et al., 2001). 6MP is structurally related to
endogenous purine bases such as adenine, guanine and hypoxanthine and hence is
metabolized by enzyme systems and pathways that metabolize endogenous purines
(Aarbakke et al., 1997). Phosphoribosylation, oxidation and methylation are the major
metabolic pathways of 6MP metabolism (Figure 1). Phosphoribosylation is an anabolic
pathway that results in the production of active metabolites that exert the anti tumor effect of
6MP by interfering with purine ribonucleotide synthesis. As opposed to phosphoribosylation,
oxidation and methylation are catabolic pathways that produce inactive metabolites. It has
been known that 6MP is converted to methylmercaptopurine (MeMP) by the action of
thiopurine methyl-transferase by a pathway that is almost exclusive for thiopurines
(Giverhaug et al., 1999). Oxidative metabolism of 6MP results in 6-thiouric acid (6TUA), 6-
thioxanthine (6TX), 8-oxo-6-mercaptopurine (8-oxo-6MP) and 6-methylmercapto-8-
hydroxypurine (6Me-8OH-MP) in vivo (Keuzenkamp-Jansen et al., 1996; Rowland et al.,
1999). There is contradictory evidence on whether 6MP is converted to 6TUA via 6TX or 8-
oxo-6MP in vivo. Early pharmacokinetic studies revealed that this drug was initially oxidized
to 8-oxo-6-mercaptopurine before being converted to 6-thiouric acid (Bergmann and Ungar,
1960; Elion, 1967; Van Scoik et al., 1985). However, Zimm et al. identified 6-thioxanthine in
urine of patients dosed with 6MP and proposed that this metabolite could also be an
intermediate in the formation of 6-thiouric acid (Zimm et al., 1984). Human xanthine oxidase
(XO) and aldehyde oxidase (AO) are very closely related molybdoflavoenzymes that have a
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high degree of amino acid sequence identity, require the same cofactors (Garattini et al.,
2003) and share a similar mechanism of action (Alfaro and Jones, 2008). However, they still
differ remarkably in their substrate specificities (Garattini and Terao, 2012). Conversion of
6MP to 6TUA has been attributed to the activity of these molybdoflavoenzymes. 6MP has a
low oral bioavailability because of extensive first pass metabolism by hepatic and intestinal
enzymes. It is believed that the drug is rapidly oxidized to its major in vivo metabolite, 6-
thiouric acid (6TUA) by the action of XO in the liver and intestine. Administration of 6MP
along with XO inhibitors have resulted in an increase in the bioavailability of this drug (Balis
et al., 1987; Giverhaug et al., 1999). Apart from this, 6MP is also converted to 6TUA by calf
liver XO, bovine milk XO (Krenitsky et al., 1972) and rabbit liver AO (Hall and Krenitsky,
1986). However, the contribution of AO/XO in the conversion of 6MP to its intermediate and
subsequently to 6TUA in humans is largely unknown. Evidence by Rashidi et al suggests that
6MP is sequentially metabolized to produce 6TUA through the intermediate metabolite, 6TX
in partially purified guinea pig liver. Rashidi et al also demonstrated that 6MP is metabolized
to 6TX exclusively by XO and subsequently converted to 6TUA by a concerted action of XO
and AO (Rashidi et al., 2007). One caveat to bear in mind is that all these in vitro
experiments have been performed using AO/XO from non-human sources. Major species
differences have already been reported for several compounds metabolized by AO and hence
extrapolation of pharmacokinetics data from other mammalian species to human is not
advisable (Choughule et al., 2013; Dalvie et al., 2013). Moreover, mammalian XOR exists in
two interconvertible forms; xanthine oxidase (XO) and xanthine dehydrogenase (XDH).
XDH preferentially reduces NAD+ and predominates in vivo whilst XO cannot reduce NAD+
and prefers molecular oxygen as an electron acceptor (Harrison, 2002). It has already been
established that XO plays a role in the metabolism of 6MP. However, the contribution of
XDH to the metabolism of this thiopurine drug still remains largely unknown.
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To understand the in vitro oxidative metabolism of this well established anti-cancer drug, we
investigated the in vitro metabolism of 6MP to 6TUA via the 6TX intermediate in pooled
human liver cytosol. The role of XO and AO were explored using specific inhibitors
raloxifene and febuxostat. We confirmed the roles by using purified human AO, and E. coli
lysate containing expressed recombinant human XO. Finally, we assessed the metabolism in
the presence and absence of NAD+ to determine the role of XDH to the overall metabolism of
6MP and 6TX.
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MATERIALS AND METHODS
Materials
6-mercaptopurine was purchased from Sigma-Aldrich (St. Louis, MO) whereas 6-
Thioxanthine and 6-Thiouric acid were purchased from Toronto Research Chemicals
(Toronto, Ontario, Canada). Febuxostat was kindly donated by Dr. Eric Kelley from
University of Pittsburgh (Pittsburgh, PA). Raloxifene was purchased from Enzo Life
Sciences (Farmingdale, NY). Sequence grade trypsin was acquired from Promega (Madison,
WI) Pooled mixed gender human liver samples (n=8) devoid of allopurinol were obtained
from St Jude’s Children’s Hospital human liver bank (Barr et al., 2014). The samples were
prepared and stored as described in (Barr et al., 2014). Table 1 contains information on the
demographics of the liver samples used to prepare cytosol. Plasmid pTrc99A containing gene
encoding for human XO was kindly provided by Tomohiro Matsumura from Nippon Medical
School (Tokyo, Japan).
Expression of Recombinant Human XOR in TP1000 Cells
Human XOR containing plasmid DNA (10ng ) was transformed into TP1000 cells (a gift
from John Enemark’s laboratory, University of Arizona). Transformants were plated on LB
agar plates containing 100µg/ml ampicillin and grown overnight at 37°C. A single colony
was used to inoculate 100 ml of LB broth containing 100µg/ml ampicillin. Cell stocks were
prepared in 15% glycerol and stored at -80°C until further use.
For expression of human XOR, glycerol stocks of TP1000 cells containing plasmid
pRTC99A encoding human XOR were plated onto LB agarose plates containing 100µg/ml
ampicillin. A single colony was picked and grown overnight at 30°C in 100ml LB broth
containing 100µg/ml ampicillin and 50µM sodium molybdate. A 10 ml aliquot of the culture
was used to inoculate 500 ml fresh LB broth with 100µg/ml ampicillin and 50µM sodium
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molybdate. Cultures were grown at 37°C and 250 rpm for 2 hours until an absorbance of 1 at
600nm was reached. Isopropyl β-D-thiogalactoside (IPTG) at a final concentration of 1mM
was used to induce cells which were then allowed to continue growing at room temperature
for 24 hours at 150rpm. Cells were harvested by centrifugation at 3500g at 4°C for 30 min in
a swinging bucket rotor (Allegra 6R centrifuge, Beckman Coulter, Pasadena, CA). Cell paste
was collected and resuspended in equal volumes of 1g of paste to 1ml of buffer (100mM
potassium phosphate buffer, pH 7.4) Cell suspension was lysed by adding lysozyme
(5mg/ml), MgCl2 (28.6μg/ml), RNase and DNase (10μg/ml each) followed by incubation at
4°C for 60 min. The cell suspension was then disrupted by sonication and subsequently
subjected to centrifugation at 100,000g for 40 min at 4°C. Supernatant containing
recombinant human XO was collected and stored at -80°C until further use.
Expression of Human AOX1 and Subsequent Protein Purification
The method of human AOX1 gene expression and purification previously developed in our
laboratory has been used in this study for the preparation of purified human AO (Alfaro et al.,
2009). Human AOX1 was overexpressed as a fusion protein with an N-terminal hexa-His tag
in TP1000 cells. Partial purification was achieved by lysing the cells and passing the lysate
through a 1ml HiTrap Chelating HP column (GE Healthcare, Little Chalfont,
Buckinghamshire, UK). The purified protein was subsequently dialyzed into 100mM
potassium phosphate buffer, pH 7.4, and snap frozen in liquid nitrogen and stored at -80°C.
Quantitation of Purified AO by HPLC-ESI-MS/MS
The amount of AO in purified samples was quantified using a technique previously
developed in our laboratory (Barr et al., 2013). A deuterium labeled synthetic peptide
standard was used to determine the amount of AO in purified samples. 25μl purified AO
sample (1.5 μM) was mixed with an equal volume of denaturing solution containing 8M Urea
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and 2mM DTT and incubated at 60°C for 60 minutes. Sodium bicarbonate buffer (25mM, pH
8.4) containing 100nM internal standard peptide was added to the mixture such that the final
reaction volume was 250μl. 10μl of 20μg trypsin was added to the reaction mixture and
incubated for 15hours at 37°C. The reaction was quenched by adding 50µl of 50% v/v
trifluroacetic acid (TFA) in water to 200µl of reaction mixture. The samples were vortexed
and centrifuged at 1460g for 10 minutes and analyzed using LC-MS/MS. The native peptide
and hence the amount of purified AO in a sample was quantified by comparing its MS peak
area to the peak area of the IS peptide of a known concentration.
Incubation Conditions
The incubation mixture consisted of substrates 6MP or 6TX at a final concentration in the
range of 10-1000µM and 0.1-200µM respectively in 25mM potassium phosphate buffer (pH
7.4) containing 0.1mM EDTA. The final concentration of dimethyl sulfoxide (DMSO) was
0.5% (v/v). For the inhibition experiments, a substrate concentration of 100µM was used.
Inhibitors, raloxifene and febuxostat were used at a final concentration of 1μM. The substrate
and inhibitor stocks were made up such that the total concentration of DMSO in the reaction
mixture was 1.5% (v/v). All incubations for 6TX and 6TUA formation were allowed to
proceed at 37°C for 15 and 30 minutes respectively. The formation of 6TX was observed to
be linear with respect to time for 15 minutes and 6TUA for more than 30 minutes. The
reaction was initiated by addition of pooled HLC at a final concentration of 0.5 or 1 mg/ml
and a known concentration of purified human AO and human XO lysate. The final reaction
volume was 80µl. The reaction was quenched by the addition of 20µl 1M formic acid
containing 1µM of 3, 5 dibromo-4-hydroxybenzoic acid as internal standard. The samples
were then centrifuged at 5000rpm for 10 minutes in an Eppendorf centrifuge 5415D and the
supernatant was collected for analysis.
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HPLC-MS/MS Assays
The samples were analyzed using 1100 series high-performance liquid chromatography
system (Agilent Technologies, Santa Clara, CA) coupled to a API4000 tandem mass
spectrometry system (Applied Biosystems/MDS Sciex, Foster City, CA) on a turbospray ESI
interface operating in the negative ion mode. Chromatography was performed on a Zorbax
Eclipse XDB-C8 column (4.6 x 150mm; 5µm; Agilent Technologies, Santa Clara, CA).
Mobile phase A consisted of 0.05% formic acid and 0.2% acetic acid in water and mobile
phase B contained 90% acetonitrile, 9.9% water and 0.1% formic acid. The column was first
equilibrated with 99% mobile phase A for 3 minutes. Chromatographic separation was
achieved using a linear gradient to 25% mobile phase A over the next 1.5minutes. Mobile
phase A was then held constant for the next 1.5 minutes before a linear gradient back to 99%
mobile phase A was achieved over a period of 1 minute. Finally, the column was
requilibrated to starting conditions over the next one minute. The entire chromatographic
assay was performed over 8 minutes per sample with a constant flow rate of 800µl/minute.
The retention times for 6-thioxanthine, 6-thiouric acid and the internal standard were 6.2, 4.5
and 6.8 minutes respectively (See supplemental figure 1). The optimized mass spectrometer
tune parameters for 6TX and 6TUA were as follows: collision gas,10; curtain gas, 30; ion
source gas 1, 50; ion source gas 2, 30; ion spray voltage, 4500; desolvation temperature, 450;
declustering potential, 70; entrance potential, 15; collision energy, 30; collision cell exit
potential, 2. The analytes, 6TX, 6TUA and the internal standard were detected using multiple
reaction monitoring mode by monitoring the m/z transition from 167.1 to 133.4, 183.0 to
140.2 and 294.8 to 251 respectively. Quantitation of the product was achieved by comparison
to a standard curve ranging from 1nM to 10µM for 6TX and 0.1 to 10 µM for 6TUA.
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Data Analysis:
Experimental data were fitted to appropriate non-linear regression models using GraphPad
Prism (version 4.03; GraphPad Software Inc., San Diego, CA).
Michaelis-Menten model:
� � ��������� � ���
Substrate Inhibition model:
� � ��������� � ��� �1 � �����
where V is the reaction velocity, [S] is the substrate concentration, Vmax is the maximum
reaction velocity, Km is the Michaelis-Menten constant and Ki is the inhibition constant for
the substrate.
Visual inspection of the velocity curve inflection (Figure 3a and 5a), as well as the biphasic
nature of the kinetic displayed by Eadie-Hofstee plot (Figures 3ai), led us to fit the data to the
equation for two or more enzymes catalyzing the same reaction. This kinetic approach
considers the total velocity as the sum of the contribution by each enzyme (Segel, 1993)
� � ����������������� � �������������
����
Here, Vmax1 is the lower Vmax, Vmax2 is the higher Vmax, Km1 is the lower Km and Km2 is the
higher Km. Since non-linear regression methods failed to converge, Lineweaver-Burk plots
were used to estimate the kinetic constants Vmax and Km for biphasic reactions. Reciprocal
plots generated using data points from the region below the first inflection of the biphasic
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curve were used to obtain Km1 and Vmax1 and points above the first inflection were used to
predict Km2 and Vmax2.
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RESULTS AND DISCUSSION
Metabolism of 6MP to 6TUA by the molybdenum hydroxylases requires two sequential
oxidations. Evidence in the literature suggests that 6MP can form 6TUA either via 6TX or 8-
OH-6MP as an intermediate in vivo in humans but the enzymes responsible for the reactions
are not known (Bergmann and Ungar, 1960; Zimm et al., 1984; Van Scoik et al., 1985) (See
Figure 1). Studies using cow XO and guinea pig AO concluded that oxidation to the 6TX
intermediate was mediated solely by XO. The second step was found to be mediated by both
guinea pig AO and cow XO (Rashidi et al., 2007). Given that many studies have shown that
animal models are poor predictor of molybdenum oxidase activity we decided to explore the
in vitro metabolism with human XO and AO (Sahi et al., 2008; Choughule et al., 2013;
Dalvie et al., 2013).
We initially explored the time-course of 6MP metabolism by monitoring the production of
the intermediate 6TX and the final product 6TUA in pooled human liver cytosol. The results
are shown in Figure 2. The intermediate 6TX is formed at a relatively linear rate for almost
25 minutes. Between 25 and 40 minutes the intermediate reaches steady state concentrations.
The final product is formed after an initial lag of about 25 to 40 minutes and produced in a
linear fashion after the intermediate 6TX reaches steady-state. These results are consistent
with sequential oxidation with release of the intermediate prior to the second step. Thus, the
intermediate 6TX must rebind to the enzyme and does not simply reorient in the active site.
Due to the unavailability of the 8-OH-6MP metabolite standard, it was not possible to
investigate the contribution of this intermediate. However, a recent crystallography study has
shown that both 6MP and hypoxanthine bind in two orientations which could lead to either
oxidation on the 5-membered or 6-membered ring of either substrate (Cao et al., 2010).
While 6MP metabolism was not explored, this study concluded that xanthine (oxidation on
the 6-membered ring) was the major intermediate of hypoxanthine oxidation and that
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oxidation on the 5-membered ring was not observed. Thus, the formation of 6TX (oxidation
on the 6-membered ring) is consistent with the results for hypoxanthine oxidation.
Furthermore, the correlation between steady-state 6TX levels and 6TUA linear rates support
that 6TX is the major intermediate, or that 6TX and 8-OH-6MP are formed with nearly
identical kinetics.
With 6TX established as an intermediate in the formation of 6TUA, the next step was to
determine the kinetics of the 6TX formation. Figure 3a shows that biphasic kinetics was
observed for 6TX formation. The Eadie-Hofstee plot shown in Figure 3ai, also displays a
biphasic profile. Biphasic profiles are generally a result of a single enzyme in possession of a
low and a high affinity catalytic binding site or two distinct enzymes involved in the
biotransformation of a single substrate, one having low and the other having high affinity
towards the substrate (Hutzler and Tracy, 2002). After using Lineweaver-Burke plots to fit
the data (Figure 3aii), Vmax1/Vmax2 and Km1/Km2 were estimated to be 0.08/0.2 nmol/min/mg
and 88/500µM respectively. Thus, one high affinity enzyme/active site and one high capacity
enzyme/active site is involved in metabolism to the 6TX intermediate but the identity of the
specific enzymes are unknown.
Specific inhibitors were used to address whether both AO and XO are involved in 6TX
production. Raloxifene and febuxostat specifically inhibit drug metabolism by AO and XO
respectively at concentrations up to 1μM. (Obach, 2004; Barr and Jones, 2013; Weidert et al.,
2014). Raloxifene inhibited the formation of 6TX by approximately 39% whereas febuxostat
demonstrated 67% inhibition (See Figure 3b). When both inhibitors were used in the same
incubation they inhibited 97% of the formation of 6TX (Figure 3b). Neither raloxifene nor
febuxostat independently resulted in complete inhibition of 6TX production implying that
both AO and XO are involved in the generation of this intermediate metabolite.
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To identify the high and low Km enzyme, 6MP was incubated with either purified
recombinant human aldehyde oxidase or E. coli cell lysate containing recombinant human
xanthine oxidase. Purified human AO metabolized 6MP to 6TX and showed substrate
inhibition kinetics with a Kcat of 0.2 min-1 and a Km of 572µM (Figure 3c). The Km value of
572µM obtained here (Table 3) is not significantly different from the Km2 value of 500µM
reported for 6TX formation in human liver cytosol above and hence it is likely that AO is the
high Km enzyme associated with 6TX formation in human liver cytosol. This also means that
XO is the likely low Km enzyme associated with this reaction. E. coli lysate containing
human XO also converted 6MP to 6TX but, since the enzyme levels are very low in the
unpurified lysate, it was not possible to perform saturation kinetics and subsequently obtain a
Km for this reaction for comparison with purified human AO. Thus, it appears that unlike
guinea pig AO, human AO mediates the formation of 6TX while both human and cow XO
can also mediate this reaction.
Unlike 6TX formation, which followed biphasic kinetics, Michaelis-Menten kinetics were
observed for the conversion of the intermediate 6TX to final product 6TUA in HLC. This
indicates that a single enzyme, or more than one enzyme with similar Km values were
involved in the catalysis of this step (Figure 4a). Inhibition experiments with raloxifene
indicated that AO was not involved in the conversion of this intermediate to the final product.
Raloxifene inhibited only 9% of 6TUA formation whereas the XO inhibitor febuxostat almost
completely inhibited 6TUA formation from 6TX (Figure 4b). In addition to this, purified
human AO did not turn over 6TX to produce 6TUA but E. coli lysate containing expressed
human XO converted 6TX to 6TUA following Michaelis-Menten kinetics (Figure 4c). These
results underpin the importance of XO in the metabolism of the intermediate to the final
product and the absence of AO contribution to this step. This is again in contrast with the
6TX metabolism by guinea pig AO and cow XO (Rashidi et al., 2007) both of which catalyze
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the reaction. Interestingly human liver cytosol has a higher Vmax and a lower Km value of
3µM than the E. coli expressed enzyme (Km, 30µM) (Table 3). Given the uncertainty in the
protein concentration of XO the rate differences between the cytosolic XO and the expressed
XO cannot be explained. However, it is surprising that the Km values are different for the
two enzyme systems. One possibility is that the human liver cytosol may have a significant
amount of xanthine dehydrogenase (XDH) as well as XO.
To assess the contribution, if any, of XDH to the formation of 6TUA from 6TX we
performed kinetic experiments on human liver cytosol in the presence of NAD+. The kinetics
of 6TUA formation from 6TX in the presence of NAD+ showed Michaelis-Menten kinetics
with a doubling of Vmax from 7 to 15 nmol/min/mg relative to no NAD+ (Figure 5a). An
increase in the Km value from 3 to 8µM was observed in the presence of NAD+ (Table 2). In
short, adding NAD+ to human liver cytosol did increase the rate of the reaction for the
formation of the final product from the intermediate 6TX and can explain, at least in part, the
difference between the cytosolic and E coli expressed metabolism. This brings into question
whether XDH may also play a role in the first rate-limiting step, the production of 6TX.
In the presence of NAD+, kinetics of 6TX was still biphasic (Figure 5b) but the kinetic
parameters indicate a 2.5 fold increase in Vmax1 value, with no significant difference in the
Vmax2 value (Table 2). (This would be expected since the second Vmax value has been
assigned to AO.) Thus, it appears that XDH and XO contribute to the high affinity reaction
while AO is responsible for the high capacity reaction.
In conclusion, 6MP is converted to 6TUA via the intermediate 6TX. Three enzymes, AO,
XO and XDH, contribute to the production of the 6TX intermediate whereas only XO and
XDH are involved in the conversion of the intermediate to the final product. This is in direct
contrast with studies using non-human tissue in which XO was found to be responsible for
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the first step, and both AO and XO where responsible for the second step. This study
establishes the enzymes responsible for oxidation of 6MP, and underlines the potential
problems associated with using other species to predict the role of human molybdenum
oxidases.
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ACKNOWLEDGEMENTS
We would like to thank Dr Tomohiro Matsumura at Nippon Medical School (Tokyo, Japan)
for providing us with the plasmid pTrc99A containing the gene encoding human XO.
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AUTHORSHIP CONTRIBUTIONS
Participated in research design: Choughule, Joswig-Jones and Jones.
Conducted experiments: Choughule and Joswig-Jones.
Contributed new reagents or analytic tools: N/A
Performed data analysis: Choughule, Jones, and Barnaba.
Wrote or contributed to the writing of the manuscript: Choughule, Barnaba and Jones.
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FOOTNOTES This work was supported by a grant from the National Institutes of Health:
[GM100874] (J.P.J)).
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FIGURE LEGENDS
Figure1- Reaction scheme showing metabolites of 6-Mercaptopurine formed by
phosphoribosylation (anabolic), methylation and oxidation (catabolic) pathways.
Figure 2- Dynamics of the formation of the intermediate, 6TX and the final product, 6TUA
in human liver cytosol over a period of 150 minutes.
Figure 3- a) Biphasic curve showing the formation of the intermediate 6TX from 6MP in
human liver cytosol (HLC) in the absence of NAD+. Points represent mean and error bars
show standard error for triplicate experiments. Shown as an inset are Eadie Hofstee plot (3ai)
and Lineweaver-Burke plot (3aii) representation of the same data. Points represent an average
of triplicate experiments. b) Inhibition of 6TX formation from 100μM 6MP by AO specific
inhibitor raloxifene (1μM) and XO specific inhibitor febuxostat (1μM) in HLC. Each reaction
was performed in duplicate and values are expressed as percentage activity relative to the
activity observed in the control (No inhibitor added). Error bars represent ± SEM. c)
Saturation plot for 6MP oxidation to 6TX in purified human AO showing Kcat (min-1) versus
substrate concentration. Data are fit to a substrate inhibition model and are an average of
duplicate experiments.
Figure 4- Michaelis-Menten kinetics for the formation of the final product 6TUA from the
intermediate, 6TX in HLC (a) and lysate containing expressed human xanthine oxidase (c)
Each experiment is an average of duplicate experiments (± SEM). Inhibition of 6TUA
formation from 100μM 6TX in HLC by AO specific inhibitor raloxifene (1μM) and XO
specific inhibitor febuxostat (1μM). Values are expressed as a percentage of activity relative
to the activity observed in of the control (No inhibitor added). Data are an average of
duplicate experiments ± SEM (b)
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Figure 5- Metabolism of 6MP to form 6TX from 6MP (b) and 6TUA from 6TX (a) in the
presence (red) and absence (blue) of NAD+. HLC was used as a source of enzyme. Data
points are an average of duplicate experiments ± SEM. Shown as figure 5bi is a Lineweaver-
Burke plot of the same data used for estimation of kinetic parameters such as Vmax and Km.
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TABLES
Liver Sex Age Disease history
1 Female 69 Hepatoma
2 Male 62 Colorectal cancer, metastasis
3 Female 68 Colorectal cancer, metastasis
4 Female 63 Cholangio carcinoma
5 Female 60 Colorectal cancer, metastasis
6 Female 70 Colorectal cancer, metastasis
7 Female 24 No information given
8 Male 57 Colorectal cancer, metastasis
Table 1: Demographics of individual liver samples pooled to generate human liver cytosol
used in this study
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Reaction Source Enzyme Kinetics Vmax
(nmol/min/mg) Km (µM)
Vmax1 Vmax2 Km1 Km2
6MP -> 6TX
HLC (-NAD) Biphasic 0.08 ± 0.00
0.2 ± 0.1
88.2 ± 4.4 500 ± 188
HLC (+NAD) Biphasic 0.2 ± 0.01
0.3 ± 0.1
83.4 ± 2.9 339 ± 169
6TX -> 6TUA
HLC (-NAD) Michaelis-Menten 7.2 ± 0.2
NA 3.0 ± 0.4 NA
HLC (+NAD) Michaelis-Menten 15.5 ±
0.7 NA 8.0 ± 1.6 NA
Table 2: Enzyme kinetic parameters for the conversion of 6-Mercaptopurine (6MP) to 6-
thioxanthine (6TX) and 6-thioxanthine to 6-thiouric acid (6TUA) in pooled human liver
cytosol in the presence and absence of NAD+. Data is representative of at least duplicate
measurements ± SEM
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Reaction Source Enzyme
Kinetics
Vmax
(nmol/min/m
g)
Kcat (min-1)
Km (µM)
Ki (µM)
Vmax1 Vmax2 Km1 Km2
6MP ->
6TX
Purified human AO
Substrate Inhibition
NA NA 0.2 ± 0.0
572± 172
NA 244 ± 77
6TX->
6TUA
Human XO
containing E . coli lysate
Michaelis-Menten
NA NA NA 30 ± 2 NA NA
Table 3: Enzyme kinetic parameters for the conversion of 6-Mercaptopurine to 6-
thioxanthine by purified recombinant human aldehyde oxidase (AO) and 6-thioxanthine to 6-
thiouric acid by E .coli lysate containing recombinant human xanthine oxidase (XO). Data is
representative of at least duplicate measurements ± SEM
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